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Agents in the Management of

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Loyola University Medical Center, Maywood, Illinois-60153, USA

ABSTRACT Sepsis, a systemic inflammatory syndrome, is a response to infection and when associated with mul- tiple is termed, severe sepsis. It remains a leading cause of mortality in the critically ill. The response to the invading bacteria may be considered as a balance between proinflammatory and antiinflammatory reaction. While an inadequate proinflammatory reaction and a strong antiinflammatory response could lead to overwhelming infection and death of the patient, a strong and uncontrolled pro- inflammatory response, manifested by the release of proinflammatory mediators may lead to microvas- cular and multiple organ failure. Endotoxin triggers sepsis by releasing various mediators inc- luding tumor necrosis factor-alpha and interleukin-1(IL-1). These cytokines activate the complement and coagulation systems, release adhesion molecules, prostaglandins, leukotrienes, reactive oxygen speci- es and nitric oxide (NO). Other mediators involved in the sepsis syndrome include IL-1, IL-6 and IL-8; arachidonic acid metabolites; platelet activating factor (PAF); histamine; bradykinin; angiotensin; comp- lement components and vasoactive intestinal peptide. These proinflammatory responses are counterac- ted by IL-10. Most of the trials targeting the different mediators of proinflammatory response have failed due a lack of correct definition of sepsis. Understanding the exact pathophysiology of the disease will enable better treatment options. Targeting the coagulation system with various agents inc- luding , activated (APC), tissue factor pathway inhibitor (TFPI) is a rational appro- ach. Many clinical trials have been conducted to evaluate these agents in severe sepsis. While trials on antithrombin and TFPI were not so successful, the double-blind, placebo-controlled, phase III trial of re- combinant human activated protein C worldwide evaluation in severe sepsis (PROWESS) was success- ful, significantly decreasing mortality when compared to the placebo group. Better understanding of the pathophysiologic mechanism of severe sepsis will provide better treatment options. Combination antith- rombotic therapy may provide a multipronged approach for the treatment of severe sepsis. Key Words: Severe sepsis, Inflammatory mediators, Microvascular thrombosis, Activated protein C (APC), Thrombomodulin, Tissue factor pathway inhibitor (TFPI), Antithrombin, activatable fibri- nolytic inhibitor. Turk J Haematol 2002;19(3): 349-389

Received: 17.06.2002 Accepted: 24.06.2002 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

Background Severe sepsis is defined as sepsis associated with acute organ dysfunction, resulting from a ge- Sepsis, a major challenge in Critical Care Me- neralized proinflammatory and procoagulant res- dicine, has been defined by the American College ponse to an infection and manifested by hypoper- of Chest Physicians/Society of Critical Care Medi- fusion and perfusion abnormalities that may inclu- cine (ACCP/SCCM) Consensus Conference, as a de, but are not limited to, lactic acidosis, oliguria systemic inflammatory syndrome in response to or acute alteration of mental status[4]. With a mor- infection which, when associated with acute organ tality rate of 30-50% despite advances in critical dysfunction such as acute renal failure, is said to care, it remains the leading cause of mortality in be severe[1]. Severe sepsis, a common, expensi- the critically ill[5-8]. Of the 750.000 cases of sepsis ve and frequently fatal condition, with annual mor- which occur each year in United States, at least tality similar to acute , is es- 250.000 are fatal[9]. Approximately two-thirds of pecially common in the elderly and its incidence is the sepsis cases occur in hospitalized patients. likely to increase with the aging United States po- Although, most of the cases of sepsis are caused pulation. The increased prevalence of human im- by gram-negative or positive bacteria, it may oc- munodeficiency virus (HIV) infection contributes cur with diseases caused by fungi, Mycobacteria, to this high incidence as well[2]. Based on 1995 Rickettsia, viruses or protozoans. Factors that state hospital discharge records from seven large predispose to gram-negative sepsis include di- states with population and hospital data from the abetes mellitus, lymphoproliferative disorders, US census, the Centers for Disease Control, the burns, cirrhosis of liver, invasive procedures or Health Care Financing Administration and the devices, drug-induced neutropenic states and American Hospital association, Angus et al, iden- asplenia. However, factors predisposing to gram- tified 192.980 cases, yielding national estimates positive sepsis include vascular , indwel- of 751.000 cases (3.0 cases per 1.000 population ling mechanical devices, burns and intravenous and 2.26 cases per 100 hospital discharges). drug injections. As a complication of broad spect- They noted the incidence increased > 100 fold rum therapy, fungal infections occur with age (0.2/1000 in children to 26.2/1000 in tho- most often in immunosuppressed individuals. The se > 85 years old). Mortality was 28.6%, or increased incidence of sepsis in the United States 215.000 deaths nationally and also increased with is attributable to the aging population, increased age, from 10% in children to 38.4% in those > 85 longevity of patients with chronic infections and years old. The estimated average costs per case the relatively high frequency of sepsis in AIDS. were $ 22100 (higher for infants, nonsurvivors, in- The widespread use of antimicrobial agents, glu- tensive care unit patients, surgical patients and cocorticoids, indwelling catheters, mechanical de- patients with more organ failure) with annual total vices and are contributing costs of $ 16.7 billion nationally[2]. Martin GS et al, factors as well. evaluated sepsis trends in the US by analyzing data from the 1988 to 1998 National Hospital The response to the invading microorganism Discharge Survey and reported at the recent 67th can be considered as a balance between the pro- annual scientific meeting of the American College inflammatory and antiinflammatory reaction. A pa- of Chest Physicians that the incidence of sepsis in tient could die of an overwhelming infection, when the US increased by 23.3% during 1988-1998. the proinflammatory reaction is inadequate and They attributed the increased incidence to the HIV the antiinflammatory response is strong. Howe- epidemic and to an increase in invasive procedu- ver, a strong and uncontrolled proinflammatory res. They reported that in 1988, sepsis was diag- response, manifested by the release of proinflam- nosed in 207.9 per 100.000 hospitalized patients matory mediators may lead to organ failure. En- and increased to 256.3 cases per 100.000 hospi- dotoxin present in the cell wall of gram-negative talized patients in 1998. The increased incidence bacteria triggers sepsis by releasing various me- was observed in neonates and patients over 55 diators such as tumour necrosis factor-alpha years of age[3]. (TNF-α) and interleukin (IL-1). These cytokines

350 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

activate the complement and the coagulation sys- tissue factor, TFPI, decreased fibrinogen, and D- tems besides expressing adhesion molecules and dimer[11]. Contact activation of the intrinsic system also releases prostaglandins, leukotrienes, reacti- of coagulation by the lipopolysaccharide in the ve oxygen species and nitric oxide (NO). The ot- cell walls of the bacteria and activation of the ext- her mediators thought to be involved in the deve- rinsic sytem by the generation of tissue factor le- lopment of sepsis syndrome include IL-1, IL-6 and ad to the generation of thrombin. The thrombin IL-8; arachidonic acid metabolites, platelet activa- can activate the thrombin activatable fibrinolytic ting factor (PAF); histamine; bradykinin; angioten- inhibitor (TAFI) which could result in fibrinolytic sin; complement components; vasoactive intesti- deficit or the thrombin can combine with the nal peptide. These proinflammatory responses thrombomodulin generated and form a thrombin- are counteracted by IL-10. thrombomodulin complex which can activate pro- tein C to APC serving as an anticoagulant. In se- Most of the trials targeting the different medi- ators of proinflammatory response may have fa- vere sepsis, activation of the coagulation system iled due to the lack of a correct definition of sep- can activate the endothelial cells resulting in the sis and also due to great flucuations in the immu- potentiation of proinflammatory responses and nological status of the patient[10]. Selected targets production of inflammatory mediators including α in the treatment of sepsis include TNF-α, IL-1, en- cytokines such as TNF- and IL-1. Thus, it appe- dotoxin, adhesion molecules, complement sys- ars that there is a role for different antithrombotic tem, kallikrein-kinin system, PAF, archidonic acid drugs in the treatment of severe sepsis, there is a metabolites, NO, reactive oxygen species, inflam- role of different agents such antithrombin, TFPI, matory reaction & immunodepression associated APC, anti-Xa inhibitors, anti-IIa inhibitors, throm- with sepsis and not the least the coagulation sys- bomodulin, TAFI inhibitors etc., could be useful tem[10]. Several agents could be used to counte- therapeutic agents. Several studies have investi- ract each of these mediators. However, it would gated the role of TFPI, anti-Xa inhibitors and APC. be difficult to target all the mediators at one time Agents to counteract increased activation of co- and on the otherhand targeting one mediator at a agulation by cytokines manifested by an increased time will be too inadequate a treatment. It is very level of tissue factor have been investigated. De- important to identify some of the very important fective manifested by an increased le- mediators and then target them simulataneously vel of inhibitor-1 (PAI-1) can or in a sequential manner. Understanding the be targeted for therapeutic intervention and supp- exact pathophysiology of the disease is essential. lementing the naturally occurring In this review targeting of the coagulation system which are decreased such as APC, antithrombin with various anticoagulant agents such as antith- and TFPI, will comprise an antithrombotic regi- rombin, activated protein C (APC), tissue factor men to treat severe sepsis. pathway inhibitor (TFPI), thrombin activatable fib- TFPI is a naturally occurring protein which can rinolysis inhibitor, thrombomodulin etc., will be dis- circulate freely or bound to low and high density li- cussed at length. However, in order to provide poproteins which inhibits both the factor VIIa/TF better treatment options, targeting of the coagula- complex and F Xa as it circulates[11,12]. While tion system and fibrinolytic system together with about 10% of TFPI is bound to the lipoproteins, other crucial mediators of sepsis will be discus- 90% of it is bound to -like species on the sed. endothelial surface and is released following the Anticoagulation in Severe Sepsis adminstration of unfractionated heparin (UFH), low molecular weight heparin (LMWHs), defibroti- The presence of intravasculat thrombi and dis- de[13,14]. Endotoxin is known to increase the circu- seminated intravascular coagulation in humans lating levels of TFPI[11,12]. A phase II study sho- with severe sepsis is evident that the coagulation wed clinical benefit of using TFPI infusion in pati- system is activated[11,12]. This is manifested by ents with severe sepsis[15]. The results of a large increased levels of activated coagulation factors,

Turk J Haematol 2002;19(3):349-389 351 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

phase III (OPTIMIST) of TFPI (Tifaco- be demonstrated in these small nonrandomized gin-Chiron/Pharmacia Corporation) in severe sep- studies with low power[21,23-25]. A phase II trial on sis indicates that tifacogin did not meet the pri- APC in sepsis did not show improved survival with mary endpoint of reducing 28-day all cause mor- APC[15]. A phase III clinical trial on APC was re- tality [PRNewswire/Chiron Corporation (Nasdaq: cently stopped after enrollment of 1500 patients CHIR), Emeryville, Calif., Nov 21, 2001]. The OP- because of efficacy associated with APC the- TIMIST trial was a prospective, double-blind, pla- rapy[26]. Such promising results ensure that infu- cebo-controlled trial investigating the use of tifa- sions of APC will in the future become part of a cogin in the treatment of severe sepsis. The trial standard therapy of severe sepsis[27]. had included approximately 2000 patients from 16 ENDOGENOUS ANTICOAGULANTS IN countries who were randomized to receive either INFLAMMATION placebo or tifacogin. TFPI was thought to prevent multiple organ failure, a major cause of death in Natural anticoagulants such as antithrombin, severe sepsis. APC and TFPI can modulate the coagulation in- duced increases in the mediators of the inflamma- In animal models of severe sepsis induced by tory response. These natural anticoagulants, be- endotoxin administration, blockade of F Xa with a sides inhibiting activated coagulation factors, can F Xa inhibitor, dansyl glutamyl-glycyl-arginyl chlo- also interact with the cells that generate antiinf- romethyl ketone-treated-Xa, prevented dissemi- lammatory substances[28]. It has been demonstra- nated intravascular coagulation (DIC) without af- ted that the generation of thrombin, F Xa and the fecting the survival rates[16]. Several clinical trials tissue factor-F VIIa complex can augment acute are underway evaluating the role of F Xa inhibi- inflammatory responses. These responses could tors in severe sepsis. be due to activation of the protease activated re- APC is a natural anticoagulant that plays a key ceptors on the endothelium leading to expression role in the regulation of coagulation by se- of adhesion molecules and platelet activating fac- lectively degrading coagulation F Va and F VIIIa tor which facilitates leukocyte activation[28]. Besi- eventually inhibiting thrombin generation[17]. Pro- des, TAFI has been recently identified in platelets tein C is one of the vitamin K-dependent plasma and could be secreted upon stimulation of the pla- proteins that is activated by the thrombin-throm- telets (Mosnier, Buijtenhuis, Marx, Meijers, Bo- bomodulin complex on the surface of intact en- uma, 2000, sumitted for publication). It has also be- dothelial cells. The anticoagulant effect of APC is en recently reported that the regulation of fibrinoly- enhanced by a cofactor, protein S, another vita- sis in plasma by TAFI and protein C is dependent min K-dependent plasma protein[18]. An endothe- on the concentration of thrombomodulin[29]. Syste- lial cell protein C receptor has been identified[19]. mic deposition of fibrin leading to impaired organ Both the plasma derived and recombinant forms perfusion thereby contributing to multiple organ fa- of protein C are now available[20]. APC is formed ilure is a hallmark of severe sepsis. Since all the when protein C is cleaved by thrombin[15,21]. Se- three major natural anticoagulant pathways are de- rum APC levels are decreased in children and fective in severe sepsis and DIC, steps to restore adults with severe Meningococcemia and purpura these pathways by administering these anticoagu- fulminans[22,23]. Based on these findings several lants, coagulation inhibitor concentrates or recom- nonrandomized trials have been conducted, where binant anticoagulant factors could markedly impro- infusions of protein C at a dose of 50-100 IU/kg ve survival and reduce the rate of multiple organ fa- [30] every 6 hours were given to adults and children ilures . A clear understanding of the role of TFPI, with severe meningococcal disease and purpura TAFI, APC, thrombomodulin and interaction of fulminans[21,23-25]. These studies demonstrated TFPI in modulation of TAFI or vice versa should that protein C infusion normalized protein C le- help untangle the complex pathophysiology of se- [31] vels, increased fibrinogen levels and resolved the vere sepsis . Furthermore, antithrombin has be- DIC. However, differences in mortality could not en shown in vitro to not only increase

352 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

responses but also to inhibit endotoxin induced plays a key role in the pathophysiology of severe calcium fluxes in monocytes, and, to inhibit nucle- sepsis. Since F VIIa and F Xa can also activate ar translocation of NFkB, an important step in the the cells in the absence of thrombin, it might be generation of inflammatory response. These natu- better to inhibit the coagulation cascade at the top ral anticoagulants in some animal models have in order to limit the coagulation mediated inflam- been shown to inhibit endotoxin/Escherichia coli- matory response[28]. Natural anticoagulants have mediated leukocyte activation and to diminish ela- shown in animal models of sepsis that they not boration of TNF-α, IL-6 and IL-8. While the phase only inhibit the inflammatory response but also III clinical trial of TFPI (OPTIMIST) failed, APC show anticoagulant activities[40-42]. Inhibition of has shown promising results in a recently comple- the functions of these natural anticoagulants can ted phase III clinical trial, trials with antithrombin potentially activate the coagulation as well as inf- were not successful[32,33]. Severe sepsis has a lammatory responses[43]. It is crucial to know complex pathophysiologic mechanism, the suc- whether or there are any independent effects of cesses and failures of each of these trials have to these natural anticoagulants on the inhibition of be critically evaluated. Until an ideal treatment re- inflammatory responses other than those of co- gimen for the management of patients with seve- agulation-mediated cellular activation. re sepsis is established, each anticoagulant, even ANTITHROMBIN those that have been shown to be unsuccessful in clinical trials have to be critically evaluated in Antithrombin levels in sepsis are seen to dec- combination with other active agents. Each agent rease by 50% of normal[44]. The fact that antith- will be discussed below to evaluate its true poten- rombin can protect healthy animals from the ad- tial either alone or in combination with other verse effects of bacterial infusion prompted the agents. protocol for replacement therapy[41]. A large pha- se III clinical trial failed to demonstrate the benefi- Coagulation leads to fibrin deposition and pla- cial effects of antithrombin. Evaluation of this trial telet activation, eventually contributing to activati- to determine reasons for failure is necessary. An- on of the leukocytes. Leukocytes are found in high tithrombin binds to heparin-like proteoglycans on numbers in venous thrombi. The leukocytes and the endothelial cell surface not only facilitates in- activated platelets can form rosettes mediated by hibition of thrombin, but has also been reported to P-selectin expression on the activated plate- induce prostacyclin formation[45,46]. The reasons lets[34,35]. Prevention of this interaction between of failure of this phase III clinical trial could be the inflammatory cells and platelets resulted in inhibi- inadequate dosage of antithrombin which does tion of both arterial and venous thrombosis in ani- not form prostacyclin in adequate amounts as hig- mal models[36,37]. Activation of the endothelium her doses are required for prostacyclin synthesis. due to thrombin results in increased leukocyte ad- Secondly, perhaps there was saturation of the an- hesion due to P and E-selectin expression[34,38]. tithrombin binding to heparin-like proteoglycans Thrombin is an agonist for the formation of PAF on the endothelial cell surface. Thirdly, perhaps and the adherent neutrophils on the endothelium the other plasma proteins might have minimized are vulnerable to the action by PAF, resulting in the the protective and beneficial effects of antithrom- release of proteases and oxidants which might inc- bin. In a large clinical trial antithrombin should rease the damage to the endothelium by various again be evaluated in combination with other na- proteases and oxidants[39]. Furthermore, the F VI- tural anticoagulants. Based on this concept of Ia-TF complex and F Xa have been shown to acti- combination therapy it is necessary to understand vate cells through protease activated receptors the mechanism of action of antithrombin to evalu- thereby generating cellular responses similar to ate its true potential. Antithrombin inactivates not those initiated by thrombin activation of protease only thrombin but also F IXa, F Xa and F VIIa receptor 1. Thrombin also activates TAFI. Thus, in- bound to tissue factor. Heparin-like proteoglycans hibition of targets such as F VIIa, F Xa or thrombin present on the endothelial cell surface help acce- may suppress the inflammatory response which

Turk J Haematol 2002;19(3):349-389 353 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

lerate these reactions. At high doses, antithrom- coagulation[59]. In rabbit and baboon sepsis mo- bin may prevent coagulation-mediated activation dels, different variants of recombinant TFPI de- of cells thereby limiting expression of adhesion monstrated increased survival[60-63]. However, in molecules, cytokines and PAF[41]. Antithrombin pigs, although there was an attenuation of the res- can cause inhibition of leukocyte adhesion and al- ponse TNF-α and IL-8, there was no significant terations in vascular permeability[47-49]. Since increase in the survival[64]. Park et al. have shown bacterial toxins compete with heparin-like prote- that TFPI binds to endotoxin, thereby depressing oglycans on the endothelial cell surface for bin- the cellular responses to bacterial cell wall and ot- ding to antithrombin, it could be used to modulate her components[65]. Although, in humans, TFPI the sepsis response[50]. Antithrombin given to ba- infusion resulted in attenuation of thrombin gene- boons challenged with E. coli, were found to have ration, the initiation of fibrinolysis and release of significantly decreased levels of IL-6 and IL-8 and cytokines, TNF-α and IL-6 were not affected[66]. IL-10[51]. Antithrombin (AT) being the main inhibi- The TFPI: Xa: VIIa: TF quarternary inhibitory tor of thrombin and F X, but other serine proteina- complex blocks protease activated receptors 2[67]. ses including F IX, F XI, F XII, plasma kallikrein, Cell activation by TF-VIIa complex can trigger a) uPA, tPA and are also inactivated by upregulation of EGR-1, b) activation of the mito- AT[52]. Low AT levels in septic are predicti- gen-activated protein kinase, c) in vitro intracellu- ve of a fatal outcome[53]. Low AT could result from lar calcium flux d) in vivo expression of reactive consumption, degradation of elastase released species and adherent molecules[68-71]. The exact from neutrophils and extravascular leakage due to mechanism of action of TFPI is still to be learned. increased vascular permeability[54]. Baudo et al., Phase III trials were not successful. recently concluded from a double-blind, randomi- ACTIVATED PROTEIN C (APC) zed, multicenter study of 120 patients receiving ATIII or placebo, that AT reduces mortality only in The protein C pathway prevents microvascu- a subgroup of patients[55]. Inthorn et lar thrombosis and neonatal purpura fulminans is al, reported that prolonged treatment with AT mi- reversed by the administration of purified protein nimizes the systemic inflammatory response re- C[72,73]. Thrombin binds to thrombomodulin (TM) sulting in decrease of IL-6 in patients with severe on the vascular endothelium and results in higher sepsis. Giudici et al, reported on the results of a concentration of TM especially in the microcircu- double-blind placebo-controlled study and conclu- lation where protein C activation takes pla- ded that within 30 days of treatment with AT, an ce[74,75]. Protein C activation is enhanced by its increased survival of patients suffering from seve- binding to the endothelial cell protein receptor re sepsis was noticed[56]. (EPCR)[76-79]. It has been shown earlier that inhi- bition of protein C binding to EPCR results in a TISSUE FACTOR PATHWAY INHIBITOR 90% decrease in the ability of thrombin to activa- TFPI is a proteinase inhibitor containing three te protein C as a response to infusion of throm- kunitz type domains[57]. The first domain combi- bin[42]. The protein C and APC bind to EPCR. The nes with F VIIa and inhibits it. The second kunitz APC as long as it is bound to soluble EPCR is not type domain combines with F Xa and inhibits it. an anticoagulant since EPCR blocks APC binding The function of the this domain is not completely to lipid surfaces and perhaps also due to change understood. In normal conditions, TFPI is expres- of specificity of APC[80,81]. APC after dissociating sion is restricted to megakaryocytes, to small ca- from EPCR, binds to protein S and this complex pillary endothelium and to macrophages. TFPI inactivates F Va and F VIIa. F V serves as an ad- blood levels are reported to increase during inf- ditional cofactor in the inactivation of F VIIIa by lammation[58]. Despite a modest increase of TFPI APC[82]. in sepsis, a significantly higher concentration (per- It is very important to understand the exact haps 10 fold) of TFPI is needed to inhibit the un- mechanism of how APC inhibits inflammation. En- controlled activation of the extrinsic pathway of dotoxin interacts with CD14 facilitating signaling

354 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

through the toll receptors and generating several tion of APC by several plasma in- signals which activates the cell. APC in complex hibitors such as, α-1 antitrypsin, α-2 antiplasmin with EPCR and interacting with a cell surface re- and PAI-1[104-106]. ceptor generates signals which block calcium inf- Assay Methods for Protein C and APC lux into the cell and translocation of nuclear factor Commercial kits to measure antigenic levels kB (NFkB). APC bound to EPCR can undergo (using plasma prepared from anticoagulated blo- nuclear translocation and can modulate gene exp- od samples) and functional activity of protein C ression profiles, to enable the cell to facilitate inf- are available (using citrated plasma samples). In lammatory responses[42]. Experiments in animals both of these assays, protein C has to be conver- have demonstrated that thrombin infusion protec- ted to APC with snake venom, and the activity of ted the animals from E. coli infusion as a result of APC is measured by either an activated aPTT-ba- thrombin activating the protein C[83]. However, in sed or an amidolytic-based assay. The protein C baboons, direct infusion of APC, protected them functional activity measurements using citrated from the lethal effects of E. coli infusion[84]. Howe- plasma can be performed in automated coagulati- ver, when protein C, protein S and EPCR were on instruments. Considerable data has accumula- blocked, the infusion of E. coli infusion became a ted on the levels of protein C due to ready availa- lethal event[84-86]. In several rodent models of bility of these assay methods and instru- sepsis, APC reduced not only IL-6, IL-8 but also ments[102,103,107-109]. However, no commercial kits decreasing the levels of TNF-α in circulation and are available for measurement of APC levels, des- tissues[87-90]. Hancock et al found a binding site pite publication of several methods[98,99,110,111]. for APC on monocytes and after binding it blocks These methods involve several steps requiring the rise of intracellular calcium and other respon- several hours to several weeks to perform with no ses[91]. Inhibition of the signaling response was commercial supply of the reagents. For direct qu- consistent of protein S. APC can block endotoxin- antitative measurements of APC levels, since induced NFkB nuclear translocation[92]. Since ele- APC is irreversibly inactivated by several plasma vation in adhesion molecules and generation of serine proteases, blood samples are collected inflammatory cytokines often require NFkB nucle- with the reversible inhibitor of APC, benzamidine ar translocation, its blockade by APC administe- in addition to citrate. The blood samples are im- red to endotoxin treated animals resulted in inhibi- mediately centrifuged to collect plasma which is tion of TNF expression and decrease in leukocyte frozen at -70°C. In the presence of benzamidine, activation. Recently it has also been established APC is immunocaptured with a monoclonal anti- that APC can prevent endotoxin-induced expres- body that blocks its active site. The excess of sion of tissue factor on monocytic cell lines in an plasma and benzamidine are removed and the EPCR dependent manner[93]. While protein C has amount of APC is measured by its ability to no biological activity, APC has shown to be antith- hydrolyze the chromogenic peptide substrate rombotic, profibrinolytic and antiinflammatory[94- (amidolytic activity) generating a yellow color. Ba- 96]. Protein C, the inactive precursor of vitamin K- uer et al and Espana et al have developed met- dependent serine protease APC, circulates in he- hods which can quantitate the levels of APC indi- althy adults at a concentration of approximately rectly[110,111]. 4000-5000 ng/mL (@ 70.000 pM), whereas the circulating concentration of APC is approximately Esmon reported that in the absence of dise- 1-3 ng/mL (@ 35 pM)[97-99]. Hence, protein C is ase with normal functioning endothelium, the normally circulating in the body approximately conversion of protein C to APC by thrombin- 2000 fold higher than APC. The circulatory half-li- thrombomodulin is dependent on the circulating fe of protein C in humans is about 10 hours while levels of protein C[112]. Thrombin generation al- that of plasma derived APC or recombinantly pro- ters the relationship between protein C and APC duced APC is only about 20 minutes[100-103]. The plasma levels. Hanson et al reported that infusi- decreased half-life of APC is as a result of inhibi- on of low concentrations of thrombin in healthy

Turk J Haematol 2002;19(3):349-389 355 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

baboons increased their APC levels from a base- vels of endogenous APC in baboons administered line of 5 ng/mL to 250 to 500 ng/mL (representing with colony forming units of E.coli intraperitone- 5000% to 10.000% increase) while endogenous ally[140]. Some baboons recovered completely, protein C levels decreased by only 15% to some sustained illness for 2 weeks and some di- 30%[113]. Likewise, prothrombotic states such as ed within 48 hours of administration of E. coli. aging, F V Leiden, and localized vessel occlusion, While there was a 50% reduction in protein C le- where there is increased generation of thrombin, vels, there was a maximum of four-fold increase without generalized endothelial dysfunction, a in the levels of APC. There was no correlation bet- less than two fold increase of APC from normal ween the decrease of protein C levels, increase of baseline was observed[110,114,115]. There was a APC levels and increase of the thrombin genera- positive correlation between the increase of APC tion markers such as TAT. On the contrary, he- with markers of thrombin generation such as althy baboons infused with low doses of thrombin prothrombin fragment F1.2, Thrombin-antithrom- showed a persistent 50-100-fold increase in endo- bin (TAT) complexes or fibrinogen fragment genous APC over the baseline with a 15 to 30% A[111,116-118]. decrease in endogenous protein C levels. In severe sepsis, the generalized systemic In phase II and phase III clinical trials of seve- response as a result of infection includes, activa- re sepsis with (activated), a re- tion of inflammatory pathways, activation of co- combinant human APC, endogenous APC levels agulation pathway, impairment of fibrinolytic path- in the placebo group during the first 2-4 days of the way and the interaction of coagulation and inflam- study and majority of placebo-treated patients with matory response, leading to generalized systemic severe sepsis, did not increase above the baseli- endothelial dysfunction, microvascular thrombo- ne level of 5 ng/mL, while the remaining placebo- sis and multiple organ failure [119-121]. In severe treated patients had levels between 5-20 sepsis, 80% of the patients have protein C levels ng/mL[122]. In the double-blind, placebo-controlled which are below the normal limits[122-125]. Low phase III clinical trial of recombinant human acti- protein C levels in sepsis relates to poor progno- vated protein C Worldwide Evaluation in Severe sis[103,109,126]. It was thought that low protein C le- Sepsis (PROWESS), the levels of APC in the pla- vels in severe sepsis were due to increased con- cebo-treated arm was similar to the placebo-tre- version of protein C to APC which has a much ated arm of the phase II study[124]. In the drotre- shorter circulatory half-life, leading to consumpti- cogin alfa treatment group with infusion for 96 ho- on of protein C. It was also assumed that if circu- urs, patients showed a 20-fold increase in the lating levels of protein C were restored to normal APC levels over the baseline and a significantly by infusion of exogenous protein C, there could decreased mortality when compared to the place- be a reduction of morbidity and mortality from bo group was observed[124]. sepsis. This formed the basis of a number of clini- ROLE of THROMBOMODULIN in cal trials and an ongoing small, placebo-controlled SEVERE SEPSIS trial of severe sepsis, where exogenous protein C was administered to restore the normal circulating It has been shown in in vitro experiments that levels of protein C, with the assumption that the endotoxin and TNF-α can decrease endothelial vasculature in patients with severe sepsis could surface thrombomodulin by decreasing synthesis adequately convert protein C to APC[127-139]. Only or increased degradation [141-143]. Endothelial sur- recently, the data started emerging on the levels face thrombomodulin may be cleaved and rele- of APC in experimental animals and humans ased in the circulation is soluble thrombomodu- which provides rationale to support the hypothesis lin[144]. Boffa et al reported on correlation betwe- of protein C replacement therapy. Recent data en increase in circulatory soluble thrombomodulin show quite the opposite that treatment with prote- in diseases with endothelial dysfunction[145]. Alt- in C may not be appropriate in patients with seve- hough elevation of soluble thrombomodulin has re sepsis. Taylor et al, recently, measured the le- been demonstrated in animal models of sepsis

356 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

and also in patients with sepsis by Dhainaut JF hydrolyzes C-terminal peptide bonds, and upon (unpublished), the reduction in endothelial surface activation by thrombin, downregulates fibrinolysis. thrombomodulin in animal models of sepsis has The carboxypeptidase activity is not present in not been demonstrated[140,146-153]. The EPCR plasma but appears only after clotting of blood. augments the conversion of protein C to APC via TAFI is an inactive zymogenic form of carboxy- thrombin-thrombomodulin as shown in in vitro ex- peptidase and the active enzyme is designated as periments. Endothelial surface levels of thrombo- TAFIa. TAFI is synthesized in the liver and cir- modulin and EPCR were reduced in skin biopsy culates in the plasma at a concentration of 4- samples from in a majority of patients with menin- 15 μg/mL[165,166]. Activation of TAFI by trypsin, gococcal septicaemia. This suggests that in pati- plasmin, thrombin or meizothrombin occurs by a ents with severe sepsis, there is insufficient en- cleavage at Arg-92. Although, sufficient proof is dothelial surface thrombomodulin and EPCR for lacking TAFI is suggested to circulate in a comp- conversion of protein C to APC[154]. Faust SN and lex with plasminogen[167]. Activation of TAFI re- colleagues are evaluating the endogenous levels duces the affinity for Glu- and Lys-plasminogen of APC in meningococcemic patients treated with by approximately 10-fold[168]. It has also been protein C[155]. This study would establish whether observed that a2-antiplasmin and e-amino capro- or not the decrease in endothelial surface throm- ic acid reduced TAFI binding to plasminogen[168]. bomodulin and EPCR results in impairment of con- TAFIa is also inhibited by EDTA, 2-mercaptoetha- version of protein C to APC. nol, peptide inhibitor from a leech, Hirudo medici- [169-171] INTERACTIVE ROLE of nalis . TAFIa because of its molecular THROMBIN-THROMBOMODULIN mass could easily be eliminated however, it rema- COMPLEX, TFPI, PROTEIN C, APC, ins in the circulation in a noncovalent complex α [172] TAFI and FIBRINOLYTIC PATHWAYS with -2 macroglobulin . The half-life of TAFIa in SEVERE SEPSIS is 10 minutes and increases with decreasing tem- peratures and is stable at 0°C[170]. Its stability is Tissue factor induced thrombin generation is achieved also by e-amino caproic acid and hepa- downregulated by TFPI and the functional protein rin[168,173,174]. TAFIa including its mutant form are [156] C pathway . Thrombin-TM complex links co- inactivated by thrombin-thrombomodulin[174]. TA- agulation with the fibrinolysis by thrombin activa- FI expression is influenced by inflammatory res- [157,158] table fibrinolysis inhibitor (TAFI) . In severe ponse in the body. The human TAFI cDNA has be- septic state increased thrombin and reduced APC en isolated and characterized[175]. TAFI was also inhibition of thrombin generation, leading to incre- expressed upon stimulation of the platelets (Mos- ased thrombin levels promote TAFI activity the- nier, Buijtenhuis, Marx, Meijers, Bouma, 2000, [159] reby inhibiting fibrinolysis . When the TM le- Submitted for publication). TAFI might protect the vels are increased the TAFI activity is reduced clot in early stages by increased levels of TAFI du- and when the TM levels are decreased the TAFI ring platelet plug formation. TAFIa inhibits fibrinoly- activity is promoted and there is more fibrinolytic sis by cleaving carboxy-terminal lysine residues deficit[160]. Protein C also combines with PAI-1 to from fibrin, limiting the formation of plasmin[176- prevent inhibition of fibrinolysis. In sepsis there is 178]. While Eaton et al identified TAFI as a conta- reduced protein C/APC activity. That formed the minant during the purification of α2-antiplasmin, basis of administering protein C to patients with Bajzar et al isolated the protein from plasma in se- meningococcal septicaemia where changes in TM arch of something which provides an explanation and EPCR results in purpura fulminans[161-163]. for the profibrinolytic effect of APC[175,176]. The ro- Maruyama demonstrated in rodent and primate le of TAFI in fibrinolysis involves activation of TA- models of TF-induced DIC that recombinant so- FI to TAFIa by plasmin and inactivation of plasmin luble TM may prevent DIC even when the AT le- by TAFIa and inactivation of TAFIa by plas- vels are low[164]. min[173,178]. TAFI is a carboxypeptidase, an enzyme that

Turk J Haematol 2002;19(3):349-389 357 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

The role of TAFI in inflammatory disease was by FDA, the physician has to carefully select suggested when it was identifies as an acute pha- among the different kinds of antithrombotic drugs se reactant[179,180]. Thrombin at high concentrati- available. Carefully conducted clinical trials for the ons not only increase TAFI generation but also other emerging antithrombotic drugs would help increased formation of TAFIa. Increased TAFI le- evaluate their potential in the management of se- vels during inflammation results in increased vere sepsis. Different classes of antithrombotic prothrombotic and antifibrinolytic state seen in agents will be discussed for they definitely have a DIC. Increased levels of soluble TM seen in DIC potential to be used in severe sepsis, to intervene stimulate the TAFI activation[181]. Hackeng et al and inhibit various steps of the coagulation casca- demonstrated the endotoxin-induced downregula- de. Further understanding of the pathophysiology tion of protein C mRNA[182]. Endotoxin also down- of severe sepsis will unfold the need for specific regulates protein C antigen, which is decreased kinds of antithrombotic, antiplatelet or antiinflam- progressively during early stages of DIC in hu- matory and thrombolytic agents. mans suggesting decreased inhibition of TAFI ac- NEW ANTICOAGULANT DRUGS tivation by protein C[165,183]. Increased TAFI levels during inflammation can either inactivate the inf- Inhibition of thrombogenesis is focused on in- hibiting thrombin, preventing thrombin generation lammatory mediators such as C3a and C5a in or- or inhibiting initiation of coagulation. Drug-deve- der to reduce susceptibility to septic shock or ca- lopment strategies involve inactivation of targeted use increased inhibition of fibrinolysis as DIC prog- coagulation factors such as thrombin, F Xa, F IXa, resses. Further studies are needed to demonstra- F VIIa/TF complex and enhancing endogenous te which mechanism functions at which particular anticoagulant pathways or promoting fibrinolysis. time. The role of antithrombin drugs needs to be Thrombin generation is important in arterial and established as they could cause decreased TAFI thrombotic disorders such as acute coronary levels resulting in decreased inhibition of fibrinoly- syndromes. It is reported that tissue factor mRNA sis. Besides the antithrombin drugs, the role of F within atherosclerotic plaques is increased com- Xa inhibitors in inhibiting F Xa at a higher step in pared with the normal arterial wall and is upregu- the coagulation cascade so as to block the gene- lated after vascular injury[184,185]. Thrombin gene- ration of thrombin have to be evaluated. ration is a trigger for initiation and furt- ROLE of OTHER ANTICOAGULANTS in her growth. Teitl and Rosenberg have earlier re- SEVERE SEPSIS ported that arterial thrombi express F Xa and F Va activity which is protected from inhibition medi- As discussed earlier, severe sepsis is mani- ated by antithrombin-dependent mechanisms[186]. fested by microvascular thrombosis resulting in In most pathologic conditions endogenous throm- multiple organ failure. This is a serious challenge bin-mediated platelet activation precedes proth- to a coagulationist despite the availability of sco- rombinase complex assembly as reported by Mi- res of antithrombotic agents to prevent the forma- letich et al[187]. Thus inhibition of thrombin and its tion of new clots; antiplatelet agents to counteract generation is one of the key targets for develop- future platelet plug formation and thrombolytic ment of new anticoagulant drugs (Table 1). Vari- drugs to lyse the prevalent clots. Although clinical ous anticoagulant drugs in different phases of cli- trials have been performed on very select antith- nical development are mentioned in Tables 2-6. rombotic agents such as antithrombin, TFPI, The scope of the new anticoagulant drugs and thrombomodulin and APC, each antithrombotic, their characteristics are mentioned in Tables 7-14. antiplatelet and thrombolytic agent has a potenti- al role to play in the management of severe sep- THROMBIN INHIBITORS sis. Each class of antithrombotic, antiplatelet and Thrombin can be inhibited indirectly by endo- thrombolytic agents have certain unique advanta- genous antithrombin or heparin cofactor II or di- ges over other classes of respective kinds of rectly by drugs that bind to thrombin thereby pre- agents. For the drugs which are already approved venting its interaction with substrates.

358 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

INDIRECT THROMBIN INHIBITORS fect-this has not been confirmed. Heparin, the most widely used intravenous 9. Heparin has a narrow therapeutic window and subcutaneous anticoagulant is a glycosami- and laboratory is required. noglycan composed of a mixture of polysacchari- The major thrust of these limitations of UFH de (14-100 disaccharide units) and has the mean led to the development of LMWHs and other molecular weight of 15.000 daltons. It combines drugs. There are several advantages of LMWHs with antithrombin (AT) causing a conformational over UFH including better bioavailability through change in its active center, and accelerates the subcutaneous administration, lower incidence of formation of thrombin-AT complexes several tho- HIT or HITTS, predictable anticoagulant respon- usandfold. Once thrombin is neutralized, heparin se, and higher antithrombotic but lower hemorrha- is released from the complex and combines with gic potential. another antithrombin molecule. Heparin with mo- re than 24 disaccharide units inhibits thrombin The LMWHs, obtained through chemical or through the interaction with AT III and with hepa- enzymatic depolymerization of the benzylic esters rin co F II. However, with fewer than 18 of porcine intestinal mucosal heparin have a par- tial effect on thrombin but mainly inhibit F Xa. He- disaccharide units cannot adequately bind throm- parins augment the activity of AT and neutralizes bin and antithrombin simultaneously. The major li- the activated forms of coagulation F X, F II, F XII, mitations of unfractionated heparin (UFH) besides F XI, F IX and TF-VIIa complex. Despite the limi- bleeding, osteoporosis and alopecia include the tations of UFH, it continues to be used since its following. complete potential is still to be unravelled. UFH 1. Heparin is an indirect thrombin inhibitor and and LMWHs are used for the prophylaxis and tre- requires antithrombin for its action. atment of venous thrombosis and as adjuncts to antiplatelet drugs and thrombolytic drugs for the 2. Heparin's anticoagulant kinetics is initially treatment of Acute Coronary Syndrome (ACS). nonlinear because of binding to different recep- Since monitoring of LMWHs is not considered ne- tors and plasma proteins; the dose of heparin to cessary, they may be used for out-of-hospital treat- saturate these recptors varies among the individu- ment[188]. At prophylactic dosages, LMWHs can- als. not be monitored by the aPTT or any other clotting 3. Patients may show heparin resistance be- test. At higher dosages of LMWHs the activated cause of limited antithrombin levels or availability. clotting time (ACT) is sensitive. The amidolytic The platelet F IV released from activated platelets AXa assay has sufficient sensitivity but is availab- interferes with the binding of heparin to antithrom- le for research and in specialized coagulation la- bin. boratories. 4. Heparin cannot inactivate clot-bound LMWHs are gradually replacing UFH for the thrombin. treatment of venous thrombosis. LMWHs are indi- cated for: 5. F Xa in the prothrombinase complex is al- so inaccessible to heparin for neutralization. 1. For prevention of DVT which may lead to pulmonary embolism. 6. Heparin can induce platelet aggregation, possible by generating A2 and by 2. In patients undergoing hip replacement sur- potentiating platelet response to adenosine dip- gery, during and following hospitalization. hosphate (ADP) and epinephrine. 3. In patients undergoing knee replacement . 7. Heparin-induced (HIT) and thrombosis syndrome (HITTS) develop in so- 4. In patients undergoing abdominal surgery me patients. who are at risk for thromboembolic complications. 8. Heparin has a circadian anticoagulant ef- 5. For inpatient treatment of acute DVT with or

Turk J Haematol 2002;19(3):349-389 359 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

without pulmonary embolism, when administered possible to give UFH and LMWHs orally by utili- in conjunction with sodium. zing synthetic amino acids such as sodium N- (8[2-hydroxybenzoyl]amino) caprylate (SNAC) 6. For the outpatient treatment of acute DVT which facilitates heparin absorption by the gut[189]. without pulmonary embolism when administered Following phase I and phase II studies, phase III in conjunction with warfarin sodium. studies are now underway to compare SNAC/he- 7. For the prevention of ischemic complicati- parin with LMWH for thromboprophylaxis in pati- ons of unstable and non-Q-wave myocar- ents undergoing elective hip or knee arthrop- dial infarction, when concurrently administered lasty[190,191]. with [188]. is a glycosaminoglycan Recent drug delivery systems have made it (GAG) that acts as an antiticoagulant by activa-

Table 1. Anticoagulant drugs launched

Agent Site Company Status Enoxaparin Xa, IIa Aventis, USA Launched Fraxiparin Xa, IIa Sanofi, France Launched Dalteparin Xa, IIa Pharmacia, Sweden Launched Heparin (Novo) Xa, IIa Novo Nordisc, DM Launched Heparin (Opocrin) Xa, IIa Opocrin, Italy Launched Reviparin Xa, IIaKnoll, GermanyLaunched Oversulfatd LMWH Xa, IIa Iketon, Pharm, Italy Launched AT Green cross IIa Green Cross, Japan Launched Antithrombin (Kabi) IIa Pharmacia, Sweden Launched IIa Mitsubishi, Kasei, Japan Launched CGP 16056 Ciba-Geigy, Swiss Launched Hoechst IIa Hoechst, Germany Launched IIa Co, USA Launched Mesoglicano Mediolanum, Italy Launched

Table 2. Anticoagulant drugs in phase III clinical trials

Agent Site Company Status H376/95 IIa Phase III Dermatan SO4, (OP) IIa Opocrin, Italy Phase III Antithrombin, CSL IIa CSL, Australia Phase III Hirudin, Ciba-Geigy IIa Ciba-Geigy, Swiss Phase III TFPI VIIa/TF Phase III APC Va,VIIIa Phase III Pentasaccharide Xa Sanofi, France Phase III

360 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

Table 3. Anticoagulant drugs in phase II clinical trials

Agent Site Company Status DX9065a Xa Daiichi, Japan Phase II IIa Astra, Sweden Phase II ART-123 Asahi Chemical, Japan Phase II MB-015 Moichida, Japan Phase II RO-46-6240 Roche, Switzerland Phase II NAP-c2 IIa Phase II SNAC-heparin Emisphere Phase II (solid formation) SNAD-heparin Emisphere (liquid formulation)

Table 4. Anticoagulant drugs in phase I clinical trials

Agent Site Company Status HV-1 Japan Energy, Japan Phase I CX-397 Japan Energy, Japan Phase I

Table 5. Anticoagulant drugs in preclinical stage

Agent Site Company Status GM-1630 Ligand Pharm’l US Preclinical GS-522 Gilead Sciences US Preclinical LEX-026 Lexin Pharm’l US Preclinical Antithrombin Genzy IIa Hoechst, Germany Preclinical Bacithrocin A Roche, Switzerland Preclinical LY-293435 Lilly, US Preclinical Hirutonins Biochem Biochem Phm, CAN Preclinical Thrombin Inhibitors Pentapharm, Swiss Preclinical SDZ-MTH-958 Novartis, Switzerland Preclinical C 186-65 COR Therap’cs US Preclinical Corthrombin compd CORVAS, US Preclinical Heparin Oral PD Xa, IIa Pharm’cal Disco, US Preclinical CVS-995 CORVAS, US Preclinical

Turk J Haematol 2002;19(3):349-389 361 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

Table 6. Anticoagulant drugs-registered or preregistered

Agent Site Company Status Antithrombin Bayer IIa Bayer, Germany Registered Dermatan Sulfate Mediolanum IIa Mediolanum, Italy Preregistered

Table 7. Scope of new anticoagulant drugs

Heparin related drugs Biotechnology Based Proteins Low molecular weight heparins Antithrombin III Medium molecular weight heparins Antithrombin III-heparin complexes High molecular weight heparins Recombinant heparin cofactor II Chemically modified heparins Glycoprotein targeting proteins & peptides Dermatans Protease-specific inhibitors Heparans Recombinant TFPI Semisynthetic heparin derivatives (Suleparoid) Peptides and related antithrombotic peptides Chemically synthesized antithrombotic Hirulogs oligosaccharides D-Me-Phe-Pro-Argderived Sulfated dextrans Synthetic hypersulfated compounds Argatroban Polyanoinic agents Inogatran Marine polysaccharides Borohydride derivatives Antiplatelet drugs Synthetic inhibitors of thrombin & related antiplatelet drugs Peptide inhibitors Platelet & related phosphodiesterase inhibitors Heterocyclic conjugates Prostanoid modulators () Nucleic acid derivatives () Eicosanoid & related drugs Others w-3 fatty acids & fish oil related products Recombinant inhibitors of thrombin Antibodies targeting membrane glycoproteins Hirudin and related proteins Peptides and proteins modulating platelet function Site-specific proteins Endothelial modulators Others Nucleic acid derivatives (Defibrotide) Polytherapy Sulfomucopolysaccharide mixtures Heparin and antiplatelet drugs 1-Deamino-8-D-arginine vasopressin (DDAVP) and Coumadin and antiplatelet drugs related peptides Thrombolytic agents and heparin Growth factor-related peptides Thrombolytic agents and antiplatelet drugs Protein digests Recombinant drugs and conjugates Vitamins Thrombolytic agents and hirudin Viscosity modulators Hirudin & Glycoprotein-targeting antibodies Synthetic and natural polymers Pentoxifyline Thrombolytic agents, hirudin and other thrombin Venoms (defibrinating agents) inhibitors Polyelectrolytes Newer drug-delivery systems and formulations Biotechnology-based products Target-specific antithrombotic drugs (antibody- Tissue type plasminogen activator & mutant directed) Hirudins, mutants and fragments Catheters and devices capable of targeted Activated protein C Drug delivery Thrombomodulin-thrombin complex

362 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

Table 8. Three generations of thrombolytic agents

First generation Second generation Recombinant tissue plasminogen activator (rtPA, , Duteplase) Anisoylated plasminogen streptokinase activator complex (APSAC, ). Single-chain Urokinase type plasminogen activator (scu-PA, prourokinase) Third generation Vampire bat salivary plasminogen activator (rPA) TNK-tPA Lanoteplase (n-PA) Staphylokinase Recombinant glycosylated plasminogen activator Thrombolytic drugs under development Antibody-targeting thrombolytic agents Polyethylene glycol-coupled thrombolytic agents Mutants and variants of plasminogen activator Recombinant chimeric plasminogen activator (Fibrolase)

Table 9. Characteristics of first generation thrombolytics

Characteristics Streptokinase Urokinase Source Gr C Streptococci Recombinant, human fetal kidney Molecular Weight (Kd) 47 35-55 Immunogenecity Yes No Mode of action Forms an activator complex Direct Plasma half-life (min) 18-23 14-20 Metabolism Hepatic Hepatic Dose 1.5 million Units 3 million Units Cost per dose $300 $2000 ting heparin cofactor II has been reported to be the prophylaxis of thromboembolism. While simi- more effective than low-dose heparin for throm- lar in structure to heparin, these agents do not boprophylaxis in cancer patients[192,193]. The de- produce any effects on platelets. Furthermore, velopmental status of glycosaminoglycan derived they are poorly absorbed after subcutaneous ad- drugs are mentioned in Table 15. Currently seve- ministration, although recently, some LMW der- ral dermatan sulfates are under development for matans are produced which unlike dermatan sul-

Turk J Haematol 2002;19(3):349-389 363 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

Table 10. Characteristics of second generation thrombolytics

Character APSAC RtPA Scu-PA() Source Gr C Streptococci plasminogen Recombinant Prodrug from a naturally anisoylated human occuring physiologic protease Molecular Weight (Kd) 131 63-70 49 Immunogenecity Yes No No Mode of action Direct Direct Direct Fibrin specificity + ++ + Plasma half-life (min) 70-120 4-6 9 Metabolism Hepatic Hepatic Hepatic Dose 30 units IV over 15 mg bolus + 90 min 20 mg bolus + 60 mg 2-5 minutes infusion infusion for 1 hour. Cost per dose $2400 $2200 $2100

Table 11. Characteristics of third generation drugs

Characteristic r-PA n-PA TNK-tPA Vampire bat PA Staphylokinase Source Recombinant, Chinese Variant of Saliva of PA of bacterial human mutant Hamster tPA-rearranging Desmodus origin-strains type PA ovary cells gene sequence rotundus of Staphylococcus aureus MW (Kd) 39 39 39 52 15.5 Immunogenecity No ? No Yes Yes Mode of action Direct Direct Direct Indirect Indirect Fibrin specificity Yes + +++ +++ +++ Plasma half-life (min) 14 37 20 170 6 Metabolism Renal Hepatic Hepatic Hepatic Hepatic Dose 20 million 120.000 U/kg 0.5 mg single 0.5 mg single 1.5 mg + 15 mg Units single bolus bolus bolus double bolus over 30 minutes

fate (Organon) are absorbed subcutaneously. He- developed for prophylactic antithrombotic use. paran sulfates have been developed as prophy- This agent produces its action via heparin cofac- lactic antithrombotic agents. These agents are ho- tor II and by inhibiting protease generation. The mogeneous and contain other chondroitin sulfa- bioavailability of this agent is better than that of tes. They bind to AT and HCII but to a lesser deg- dermatan and heparan sulfates. However, this ree than heparin, and they are weakly anticoagu- product exhibits teratogenic potential and clinical lant. Thus, large doses of heparan are needed for trials have therefore been suspended. effective antithrombotic treatment. Depolymerized A semi-synthetic sulfated pentomannan deri- heparans have better bioavailability than the nati- vative PI-88, phosphomannopentaose sulfate ve heparans. A synthetic hypersulfated lactobi- (Progen Industries, Brisbane, Australia) has been onic acid amide, aprosulate (Luitpold) has been shown to exhibit anticoagulant activity via heparin

364 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

Table 12. Classification of GPIIb/IIIa inhibitors

Structure Receptor binding Genetic name Trade name Company Monoclonal Binds to GPIIb/IIIa Abcixmab ReoPro Centocor, Lilly antibody receptor and inhibits binding of large adhesive ligands by steric blockade Peptide Competitive antagonist; EptifibatideIntegrelin COR receptor binds specifically to the Therapeutics/ antagonist fibrinogen binding site Scherring-Plough Nonpeptide Competitive antagonist; Aggrastat Merck & Co. receptor antagonist/ binds specifically to the peptidomimetic fibrinogen binding site Oral GPIIb/IIIa Competitive antagonist; Lamifiban Roche agents/ bind specifically to the Xemillofiban Searle Peptidomimetic fibrinogen binding site Roche/genentec prodrugs Orbofiban Searle Lotrafiban SK Beacham RPR-109891 Aventis Roxifiban DuPont Lefradafiban BoehIngelheim

Table 13a. Molecular and chemical characteristics of various LMWHs

LMWH Characteristics Enoxaparin Presence of 4,5 unsaturated uronic acid at nonreducing termi- nus Nadroparin Presence of 2,5-anhydro-D-mannose at reducing terminus Certoparin Presence of 2,5-anhydr-D-mannose at reducing terminus Dalteparin Presence of 2,5-anhydr-D mannose at reducing terminus Tinzaparin Presence of 4,5 unsaturated uronic acid at nonreducing Reviparin Presence of 2,5-anhydro-D-mannose at reducing terminus Ardeparin Labile glycosidic bonds cofactor II activation and TFPI release. This agent veloped for the prophylaxis of thromboembolism. is being developed as a potential antitumor agent, Some of these represent mixtures of GAGs with having the important property of simultaneously varying molecular weight profiles. Noteworthy are being potent inhibitors of in vitro angiogenesis Intimitan, Lomoparan and suleparoide which are and heparanase activity. PI-88 inhibited the pri- depolymerized heparan preparations. These mary tumor growth of the highly invasive rat mam- agents exert their antithrombotic actions via unk- mary adenocarcinoma by approximately 50%, in- nown mechanisms but are clinically very effective hibited metastases by approximately 40% and re- drugs. Additional synthetic heparinomimetics inc- duced the vascularity of tumors by approximately lude synthetic oligosaccharides with high affinity 30%. This agent is undergoing phase II clinical tri- to AT. More recently, mixed inhibitors of F Xa and als. F IIa have also been developed. Many other glycosminoglycans are being de- There have been significant developments in

Turk J Haematol 2002;19(3):349-389 365 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

Table 13b. A comparison of LMWH preparation

Agent Axa/IIa ratio USP U/mg Enoxaparin 3.8 35 Nadroparin 2.3 34 Certoparin 2.3 46 Dalteparin 2.8 53 Tinzaparin 1.9 49 Reviparin 3.4 32 Ardeparin 2.0 51 Each LMWH is different and cannot be used interchangeably. The reasons why different LMWHs are not interchange- able are as follows: 1. Due to manufacturing procedures, different products have different physical and chemical compositions. This transla- tes into the differences in biologic actions. 2. The amount of pharmacologically active (chemically active) material varies from product to product. 3. Clinical trials for specific indications on each product are carried out at optimised dosages for each product. Thus, a specific dosage used for individual products must be used. 4. Each drug is classified by the USFDA as a distinct drug and cannot be interchanged.

Table 14. Direct factor Xa inhibitors

Agent Company Chemical Source Status ANTISTATIN Merck Sharp Mexican leech Recombinant Suspended & Dohme protein (119 amino acids) YAGIN Bio-Technology Medicinal leech Animal derived Not reported General protein (85 amino acids) TAP Merck Sharp Tick protein Recombinant Preclinical & Dohme (60 amino acids) NAP-5 CORVAS Hookworm protein Recombinant Preclinical TFPI Searle/Chiron Human protein Recombinant Clinical DX-9065a Daiichi Propanoic acid derivative Synthetic Phase II SEL-2711 Selectide Pentapeptide produced Synthetic Preclinical by combinational chemistry YM-60828 Yamanouchi Synthetic Preclinical BX-807834 Berlex Peptidomimetic Synthetic Preclinical KFA-1411 Kissei Peptidomimetic Synthetic Preclinical RPR-120844 Aventis (RPR) Peptidomimetic Synthetic Preclinical INDIRECT Xa INHIBITOR SR-90107 Sanofi Oligosaccharide; requires Synthetic Phase III binding to AT

366 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

the area of nonheparin GAGs-derived products as prophylactic antithrombotic usage in patients un- antithrombotic drugs. It is no longer believed that dergoing general surgery. There have been con- a sulfomucopolysaccharide of natural origin must cerns over the safety and efficacy of the higher mo- exhibit some interaction with AT to have effective lecular weight dermatans such as MF-701 and OP- antithrombotic properties. Several agents without 435 and as a result low molecular weight derma- this interaction produce therapeutic effects on the tan preparations have been introduced. One such blood and vascular system[194-195]. Several mam- preparation is Desmin (Alfa wasserman), which malian GAG-derived drugs are currently being exhibits better bioavailability, and longer duration used in European countries as antithrombotic, an- of action than the high molecular weight derma- tilipemic and antiatherosclerotic agents[196]. The- tans, is being developed for prophylactic antith- se agents represent mixtures of native sulfomuco- rombotic use. MPS (Luitpold) represents a mixtu- polysaccharides or its derivatives obtained by de- re of mucopolysaccharides obtained from mam- polymerization and/or fractionation. With an incre- malian trachea for the treatment of joint diseases. ase in knowledge of their structure and functional Only limited data is available on the structure ac- activity, preliminary pharmacological studies were tivity relationship of this agent. This agent may ha- carried out to determine the proper indications for ve several applications as an antithrombotic individual drugs. The GAG-derived drugs are ge- agent. Some GAGS may prove to be useful as an nerally used as antithrombotic agents, however, alternative to heparin especially in HIT and several other indications such as atherosclerosis, HITTS. Some of the newer indications of these stroke, hyperlipidemia and senile dementia are agents include antiinflammatory, antiatherosclero- now being considered. A list of GAG-derived an- tic, for wound healing and as a treatment of AIDS, tithrombotic drugs are mentioned in Table 15. SP- besides other indications such as Alzheimer’s di- 54 (Hemoclar; Bene Chemical), a hypersulfated sease and as a cytoprotective agent[198-202]. He- pentosan polysulfate with structural and functional paran sulfate has been studied in DVT, chronic characteristics similar to other sulfated GGS, is a venous insufficiency and intermittent claudicati- plant (beech tree) product. on[198,201]. A pilot study was earlier completed using dermatan sulfate in acute leukemia to cont- sodium is a depolymerized mixtu- rol disseminated intravascular coagulation re of heparans, dermatans and other chondroitin (DIC)[203]. sulfates and is undergoing clinical trials for the prophylaxis of DVT after general and orthopedic DIRECT THROMBIN INHIBITORS surgery. This agent is also being used in the pre- Direct thrombin inhibitors in contrast to UFH vention of ischemic complications associated with can inhibit fibrin-bound thrombin, have more pre- stroke. It is claimed to have a better safety/effi- dictable anticoagulant response since they do not cacy ratio than heparin, such that it produces mi- bind to plasma proteins, and are not neutralized nimal antihemostatic effects at antithrombotic do- by platelet F IV[204-207].The developmental status ses[197]. MF-701 (Mediolanum Laboratories) is a of different direct thrombin inhibitors is given in heterogeneous mixture of dermatan sulfate of Table 16. mammalian mucosal origin. Currently, it is being developed for prophylaxis against DVT after ge- HIRUDIN neral and orthopedic surgery. Since the bioavaila- Hirudin, a 65 amino acid polypeptide originally bility of this agent via subcutaneous administrati- isolated from the parapharyngeal salivary glands on is rather limited, it is being administered intra- of a medicinal leech, Hirudo medicinalis, is now muscularly and several clinical trials are ongoing available through recombinant DNA techno- with this agent. Suleparoide is a widely used se- logy[208]. It is the most potent and specific inhibi- mi-synthetic GAG that has been used for the tor of thrombin known and forms a 1:1 stoichi- prophylaxis of both arterial and venous thrombo- ometric complex with this enzyme which is slowly sis. OP-435 (Opocrin Laboratories) is extracted reversible[209]. A number of derivatives and re- from bovine mucosa and is being developed for

Turk J Haematol 2002;19(3):349-389 367 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

Table 15. Glycosaminoglycan-derived antithrombotic drugs

Drug Composition Status ORG 10172 Depolymerized mixture of GAGS Ongoing clinical trials MF 701 Mixture of native and depolymerized dermatans Ongoing clinical trials Suleparoide Semi-synthetic GAG Available for various indications OP 435 Mixture of dermatans Preclinical OP 370 LMW dermatan Preclinical SP 54 Hypersulfated pentosan polysulfate Preclinical MPS Depolymerized hypersulfated Developed for animal use mixture of GAGS Sulfomucopolysaccharide Mixture of GAGS Clinically used mixture

combinant preparations are now available, inclu- approval in patients with and ding Hirugen, a synthetic C-terminal peptide frag- non-ST-elevation myocardial infarction. The ad- ment of hirudin; Hirulog (bivalirudin), a derivative vantages of hirudin over UFH are given in Table of hirugen. The various recombinant preparations 17. are, desirudin (CGP 39393); (HBW 023, Refludan (lepirudin-rDNA for injection was Refludan); Polyethyleneglycol-coupled hirudin successfully used for anticoagulation and effecti- (PEG-hirudin) obtained by conjugating recombi- vely monitored with Ecarin Clotting Time (ECT) in nant hirudin with two molecules of PEG; and albu- patients with HIT undergoing off-pump coronary min r-hirudin fused molecules. Both PEG hirudin artery revascularization[220]. and albumin r-hirudin fused molecules are known to have longer half life compared to r-hirudin. Hi- BIVALIRUDIN rudin has a plasma half-life of 40 minutes after int- Coupling of the peptides that mimic the car- ravenous administration and 120 minutes after boxyterminal of hirudin to peptides that are speci- subcutaneous administration and is cleared most- fic for inhibition of the catalytic site of thrombin (D- ly by kidneys after undergoing little hepatic meta- Phe-Pro-Arg) has led to the development of a se- bolism[210]. Recombinant hirudin has been used mi-synthetic bivalent thrombin inhibitor, bivaliru- for prophylaxis of thrombosis and thromboembo- din[221]. It is a specific thrombin inhibitor by bin- lic complications in patients with HIT and has be- ding to both catalytic site and its anion binding en approved in the USA for this specific indicati- exosite. Bivalirudin is a specific and direct inhibi- on[211-213]. Hirudin has also been used as an al- tor of free and clot-bound thrombin. The hirulog- ternate to heparin in HIT patients undergoing car- thrombin complex is transient because thrombin, diopulmonary by-pass surgery, and was found to once complexed, can slowly cleave the Arg3-Pro4 be superior to low dose heparin subcutaneous bond on the amino-terminal extension. This meta- UFH or LMWH for thromboprphylaxis in patients bolic cleavage, converting bivalirudin into a lower undergoing elective hip arthroplasty without incre- affinity inhibitor, contributes to its short half-life[222- asing the risk of bleeding[214-217]. Recombinant hi- 224]. Bivalirudin is only 20% excreted in the urine, rudin is found to be more effective than heparin in indicating that it is either extensively catabolized patients with unstable angina and non-ST-elevation by the liver or undergoes proteolysis at other si- myocardial infarction, although it increased the tes. Following phase III trials showing enhanced risk of bleeding in these patients. There was no safety of bivalirudin relative to UFH in patients un- increase in life threatening bleeding complicati- dergoing coronary angioplasty, it has been appro- ons[218,219]. Hirudin is now being considered for ved in the US for this indication[225,226]. In one pi-

368 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

lot study, angiographic patency of the culprit coro- rapy to low dose or high dose argatroban or hepa- nary artery lesion was assessed 90 and 120 mi- rin alone, excessive cardiac events were noticed nutes after the initiation of streptokinase and aspi- with low dose argatroban and this arm was drop- rin and again after 4 ± 2 days in 68 patients with ped. The rates of mortality, recurrent MI and ma- AMI[227]. In this trial bivalirudin yielded higher pa- jor bleding were no different between high dose tency rates when used in conjunction with strepto- argatroban and heparin. kinase and aspirin in the early phase of AMI. Hig- H376/95 her bivalirudin doses are unnecessary and may not be better than lower doses, suggesting the It is prodrug oral formulation of Melagatran fact that too much thrombin inhibition may actually which is currently under phase III trials for preven- be harmful. tion and treatment of venous thrombosis. It is well ARGATROBAN absorbed from the gastrointestinal system and af- ter rapid biotransformation is converted to an ac- Argatroban (Novastan) is a carboxy acid deri- tive site-directed thrombin inhibitor-melagat- vative, belonging to a class of peptidomimetics ran[231,232]. that also includes inogatran, efegatran and nap- sagatran. Argatroban has now been approved in EFEGATRAN the US as an alternate to heparin in patients with Efegatran sulfate (GYKI 14766), a tripeptide HIT. It binds covalently to the active site of throm- aldehyde (mePhe-ProArg-H), is an arginal cataly- bin[228]. Argatroban was used in one trial of 50 pa- tic-site inhibitor of thrombin and is a reversible, tients with HIT undergoing PTCA, at a dose of 350 competetive, tight-binding inhibitor[233,234]. Efe- μg/kg bolus and yielded encouraging results[229]. gatran is being evaluated in several clinical trials. In the myocardial infarction with Novastan and NAPSAGATRAN tPA (MINT) study, low and high dose argatroban yielded 90 min TIMI grade 3 flow rates approac- Napsagatran (RO-46-6240) is a cyclopropyl hing 60%[230]. The rates of bleeding were similar derivative of a novel class of thrombin inhibitors to heparin. However, Argatroban in Acue Myocar- and is a selective, potent, competetive and rever- dial Infarction (ARGAMI) II trial wherein 1200 pa- sible inhibitor of thrombin[235]. Napsagatran is cur- tients were randomized to receive fibrinolytic the- rently in phase II clinical trials for preventing pos-

Table 16. Developmental status of thrombin inhibitors

Drug Chemical nature Status

Hirudin (Refludan) Recombinant protein Alternate anticoagulant in management of HIT Hirulog (Angiomax) Synthetic bifunctional oligopeptide Approved in PTCA. Several clinical trials completed & planned Argatroban Synthetic heterocyclic derivative Phase II & III clinical development in USA; approved in USA. Aptamers DNA and RNA-derived Preclinical stage; limited Oligonucleotides with animal data available thrombin-binding domains Plasma-derived Protein and their recombinant Antithrombin III is currently used. antithrombin equivalent products HC-II is still in developmental stage Oral thrombin inhibitor Prodrug for the management of DVT Phase II and III clinical trials PI-88 Sulfated pentomannan Phase II clinical trials.

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toperative thrombosis and treating venous throm- lonal antibodies against F IX/IXa blocking F X ac- bosis. tivation by F IXa[238-240]. Antithrombotic activity in a rat model of thrombosis has been achieved uti- INOGATRAN lizing a chimeric humanized derivative of this an- Inogatran (H314/27) is a synthetic dipeptide tibody[238,239]. which selectively, rapidly and competetively binds FACTOR Xa INHIBITORS thrombin[236]. Further clinical development of ino- gatran has been stopped. The F Xa inhibitors could be classified as fol- lows: FACTOR IXa INHIBITORS Indirect Inhibitors of Factor Xa F IXa which is essential for amplification of co- agulation could be inhibited either by active site F a. Synthetic pentasaccharide (analogue of IXa inhibitors or by monoclonal antibodies direc- pentasaccharide sequence of heparin UFH and ted against F IX/IXa. LMWHs have limited ability to inhibibit platelet-bo- [241-243] Active-Site Factor IXa Blockers und F Xa . The intrinsic tenase complex assembles on Direct F Xa inhibitors: Inhibit F Xa bound to [244] the surface of the activated platelets. By compe- phospholipid surfaces and free F Xa . A list of ting with F IXa for its incorporation in the tenase various direct F Xa inhibitors is given in Table 14. complex, the active site F IXa is blocked. The a. Natural Inhibitors like Tick anticoagulant blocked F IXa inhibits clot formation in vitro and is peptide (TAP) and antistatin. shown to inhibit clot formation in coronary artery b. Synthetic inhibitors like DX9065a, YM- thrombosis in a canine model[237]. 60828, SF 303 and SK 549. Antibodies Against Factor IX/IXa INDIRECT FACTOR Xa INHIBITORS Inhibition of F IX activation in addition to bloc- Pentsaccharide produces its antithrombotic kage of F IX activity could be achieved by monoc-

Table 17. A comparison of r-Hirudin and UFH r-Hirudin Unfractionated heparin Monocomponent protein with single target Polycomponent drug with multiple sites of action (thrombin) Thrombin-mediated amplification of coagulation Thrombin and F Xa feedback amplification of is affected only under certain conditions clotting is affected No known interactions with endothelium other Significant interactions with endothelium. Both than blocking the thrombin-thrombomodulin- physical and biochemical modulation of mediated activation of protein C endothelial function Shorter half-life via IV route Short half-life via IV route. Functional bioavailability is variable and Functional bioavailability is 20-30%. LMWHs are dependent on the structure of r-hirudin better absorbed Endogenous factors (PF4, FVIII) do not alter Marked modulation by the endogenous factors. its antithrombotic action Several factors may alter the anticoagulant actions Relatively inert proteins not altered by Transformed by several enzyme systems and reduces metabolic processes its anticoagulant actions Information on cellular uptake and depo Significant cellular uptake and depo formation formation is not presently known

370 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

effect via binding to antithrombin. I t has a mole- DX9065a, YM-60828, SF-303 and SK-549 are cular weight of 1.728 daltons. It was developed in nonpeptide, low-molecular-weight, reversible inhi- 1983 to show that a five member heparin chain bitors of F Xa and are effective in various animal was the minimum saccharidic sequence needed models of thrombosis[257-260]. DX-9065a is under- for antithrombotic activity[245-247]. In various expe- going phase II clinical evaluation in patients with rimental models, it has been shown that inhibition unstable angina. of factor Xa controls excessive thrombin generati- Factor VIIa/Tissue Factor Pathway on and produces antithrombotic effect with lesser Inhibitor bleeding risk than heparin[248-250]. Recently, in in vitro experiments, it has been shown that the anti- The F VIIa/TF pathway being the initial coagu- coagulant activity of synthetic pentasaccharide lation pathway, much attention has been given in can be neutralized by Heparinase, an eliminase blocking this pathway by developing F VIIa inhibi- [251] isolated from Flavobacterium heparinum . A tors and tissue factor pathway inhibitors synthetic pentasaccharide analogue, SANORG (TFPI)[261]. 34006 has also been developed which shows a longer half-life. However, SANORG 34006 was fo- TFPI und to be resistant to heparinase I neutralizati- Tissue factor pathway inhibitor is a protein bo- on[251]. Pentasaccharide is under phase III clinical und to low density lipoproteins and high density li- evaluation for prophylaxis of venous thrombosis, poproteins and as it circulates it inhibits both the comparing LMWHs. VIIα-TF complex and F Xa. While about 10% of DIRECT INHIBITORS of FACTOR Xa TFPI is bound to the lipoproteins, 90% of it is bo- Antistatin: Antistatin, isolated and purified und to heparin like species on the endothelial sur- from the mexican leech, Haementeria officinalis, face and is released following the administration is a 119 aminoacid polypeptide with a molecular of unfractionated heparin, LMWHs, Defibrotide weight of 17.000 daltons[252]. It inhibits F Xa by and PI-88[262,263]. It has been shown that endot- forming a stable enzyme -inhibitor complex[253]. helial depletion of TFPI may contribute to “rebo- Since it has a potential to develop antibodies, it und” thrombin generation, following the sudden has been stopped for future development. cessation of unfractionated heparin[264]. TFPI has Yagin: Yagin, isolated from the medicinal le- shown to attenuate injury-induced neointimal ech, Hirudo medicinalis, is a 85 amino acid pepti- hyperplasia in Pigs and also inhibits smooth de with 50% homology with antistatin. It is a slow muscle cell migration in vitro TFPI has also shown tightbinding inhibitor of F Xa[254]. to attenuate the coagulopathy and improve survi- val in sepsis models in rabbits and baboons[265]. TICK-ANTICOAGULANT PEPTIDE (TAP) TFPI is now undergoing phase III clinical trials in TAP is originally isolated from the tick, Ornit- patients with sepsis. hodorus moubata, and now manufactured thro- NAP c2 and NAP-5 ugh recombinant DNA technology, is a 60 amino acid peptide (6.850 daltons) with the potential of These are two of the anticoagulant proteins inhibiting human F Xa by its slow and tight-binding isolated from hookworm nematode, Ancylostoma mechanism, initially forming a weaker complex caninum and NAPc2 is currently undergoing pha- and later forming a more stable enzyme comp- se II clinical trials for prevention of venous throm- [255,256] lex . TAP has been shown as an antithrom- bosis in patients with elective knee arthroplasty. botic in experimental models of venous and arte- NAPc2 binds to a noncatalytic site on F X or F Xa rial thrombosis and also showed favorable antip- and inhibit F VIIa within the F VIIa/tissue factor roliferative effects of smooth muscle cells in reste- complex has a half-life of 50 hours following sub- nosis processes. cutaneous administration[266]. It attenuates sep- SYNTHETIC FACTOR Xa INHIBITORS

Turk J Haematol 2002;19(3):349-389 371 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

sis-induced coagulopathy in laboratory animals. coagulopathy. NAP-5 inhibits F Xa and the F VII/TF complex af- Modulation of the Endogenous [267] ter prior binding to F Xa . Fibrinolytic Activity Other Antithrombotics Enhancing TAFI: It is a latent carboxypeptidae B like Endogenous Anticoagulant Activity enzyme that is activated by thrombin-thrombomo- Thrombomodulin: Thrombomodulin is an en- dulin complex, and attenuates fibrinolysis by cle- dothelial cell surface protein that forms a complex aving carboxy-terminal lysine residues from fib- with thrombin, which makes the thrombin lose its rin[282,283]. The fibrinolytic process is retarded by procoagulant property and activates protein C removal of these lysine residues which decreases thousandfold compared to free thrombin[268-270]. the plasminogen or plasmin binding to fibrin. It APC in the presence of protein S inactivates co- has been shown in dogs and rabbits that a pota- agulation F Va and F VIIIa[271,272]. Thrombomodu- to-derived carboxypeptidase B inhibitor increases lin besides playing an important role as endoge- tPA-induced [284,285]. nous regulator of coagulation on the surface of Factor XIIIa Inhibitors vascular wall, also inhibits the proteolytic action of thrombin on macromolecular substrates and inac- The Laki-Lorand F XIIIa, a thrombin-activated tivation of thrombin by antithrombin. Thrombomo- transglutaminase, crosslinks the α-and γ-chains dulin is an integral membrane glycoprotein pre- of fibrinogen to form α-polymers and γ-dimers res- sent on the vascular surface of endothelial cells of pectively. As the fibrin polymer is stabilized due to arteries, veins, capillaries and lymphatic vessels. crosslinking, it is rendered more refractory to deg- The recombinant human oluble thrombomodulin radation by plasmin[286]. It is therefore thought is now available and is found to be effective in the that inhibition of F XIIIa makes the thrombus sus- rat model of arteriovenous shunt thrombosis and ceptible to lysis. Tridegin, a peptide isolated from in disseminated intravascular coagulation models the giant Amazon leech, Haementeria ghilianii, is in mice and rats and in situations where antith- a specific F XIIIa inhibitor and has shown to en- rombin levels are reduced[273-276]. Parenteral ad- hance fibrinolysis in vitro when added prior to clot- ministration of soluble recombinant thrombomo- ting of fibrinogen[287,288]. Destabilase, a leech dulin has shown antithrombotic effects without enzyme that hydrolyzes γ-gcrosslinks also inhibits any bleeding in cancer patients[277]. F XIIIa action[289,290]. Protein C: APC is a natural anticoagulant that PAI-1 Inhibitors plays a key role in the regulation of blood coagu- Inhibition of PAI-1 which is a major physiologic lation by selectively degrading coagulation F Va inhibitor of tPA and u-PA results in increased en- and F VIIIa eventually inhibiting thrombus genera- dogenous fibrinolytic activity. PAI-1 synthesis is [278] tion . Protein C is one of the vitamin K-depen- decreased in vitro by lipid lowering drugs such as dent plasma proteins which is activated by the niacin and fibrates[291,292]. Similarly peptides that thrombin-thrombomodulin complex on the surface block PAI-1 activity are also identified which either of the intact endothelial cells. The anticoagulant prevent insertion of the reactive center loop upon effect of APC is enhanced by a cofactor, Protein cleavage by the target protease or by converting [279] S, another vitaminK plasma protein . An en- PAI-1 into a latent conformation[293,294]. Develop- dothelial cell protein C receptor has been identifi- ment of small molecule PAI-1 inhibitors, some of [280] ed earlier . The circulating plasma concentra- which may have antithrombotic activity in vivo tion of protein C is 4 mg/L. Both the plasma de- may provide a more promising alternative stra- rived and recombinant forms of protein C are now tegy[295]. available[281]. Intravenous APC has shown bene- ficial in the treatment of patients with sepsis-indu- Glycoprotein IIb/IIIa Inhibitors ced coagulopathy[280]. Currently, it is undergoing GPIIb/IIIa inhibitors block the final common phase III clinical evaluations for sepsis-induced

372 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

pathway of platelet aggregation[296]. sesed by angiography were seen. The preliminary during percutaneous coronary interventions has results of an ongoing double-blind, randomized, reduced 30-day ischemic outcomes by approxi- placebo-controlled crossover trial of abciximab mately 35-50%[297-299]. The clinical development alone or in combination with low-dose activase of peptide and peptidomimetic GPIIb/IIIa inhibitors in 26 patients with AMI, who presented within 6 have shown less consistent benefits[300-302]. The hours of symptom onset with ST-segment eleva- oral GPIIb/IIIa inhibitors have demonstrated app- tion were initially given aspirin and heparin and roximately 30% increase in mortality[303]. A safe then randomized to receive either abciximab 0.25 and effective level of GPIIb/IIIa inhibition by rapid mg/kg bolus or placebo followed by an angiogram platelet function testing will allow the optimization 60-90 minutes later. Patients were crossed over of doses in all patients. A list of GPIIb/IIIa agents and given the opposite treatment. A second angi- are mentioned in Table 12. ogram was taken 10 minutes later. Those patients in which TIMI grade 3 flow was not achieved we- The GPIIb/IIIa inhibitors can be used in com- re further randomized to receive activase 20 mg bination with the thrombolytic agents in patients or placebo. A third angiogram was performed 15 with acute myocardial infarction. Activase (Altep- minutes later. The results of the second angiog- lase, recombinant) in combination with GPIIb/IIIa ram where patients received abciximab alone, 8 inhibitors or TNKase in combination with GPIIb/II- patients had TIMI grade 0 flow, 5 patients had TI- Ia inhibitors can be used in patients with acute MI grade 1 flow, 5 patients had TIMI grade 2 flow myocardial infarction. anf 8 patients had TIMI grade 3 flow. The results The thrombi in the coronary arteries causinh of the angiogram in patients receiving activase acute myocardial infarction comprise of a platelet and placebo are not yet reported[307]. core in a fibrin-thrombin matrix. Following suc- Antman et al also reported the results from the cessful thrombolysis, the reocclusion is caused by dose-finding and dose-confirmation phases of TI- excessive platelet activation which makes the MI-14 trial which evaluated the use of thromboly- thrombi difficult to lyse. In these situations, ad- tic therapy in combination with abciximab in pati- junctive use of thrombolytic agents with GPIIb/IIIa ents with AMI[308]. inhibitors will prevent platelet activation and agg- regation[304]. Platelet binding to the walls of the TNKase, a new genetically engineered variant vessel by attachment at Ia or Ib receptors on the of tissue plasminogen activator is produced by the platelet surface. Platelet-platelet binding is as a recombinat DNA technology. TNkase is fibrin spe- result of interaction between GPIIb/IIIa receptors cific. This fibrin specificity decreases systemic ac- involving the fibrinogen and von Willebrand fac- tivation of plasminogen and the resulting break- tor[305]. It was demonstrated by Gold et al that the down of the circulating fibrinogen when compared platelet Fab fragment of the murine antibody 7E3- to a molecule lacking this feature. The ASSENT-2 F(ab)2 to GPIIb/IIIa binds tightly to the GPIIb/IIIa was a phase III randomized trial, double-blind trial receptor and inhibited platelet aggregation[306]. In that compared TNKase with Activase. Currently, TAMI-8, a nonrandomized multicenter pilot study, there is no published information on the safety and 60 patients with AMI were given activase with va- efficacy of TNKase in combination with GPIIb/IIIa ried abciximab dosages of 0.1 mg/kg, 0.15 mg/kg, inhibitors. Anticoagulants such as heparin and Vi- 0.20 mg/kg and 0.25 mg/kg given at 3, 6, and 15 taminK antagonists, acetylsalicylic acid, dipyrida- hours after a 100 mg dose of activase administe- mole and GPIIb/IIIa inhibitors may increase the red over 3 hours. Despite limitations of the study risk of bleeding if administered prior to, during or being small and not blinded, the safety profile was after TNKase therapy. similar in the abciximab and control groups. Ho- SEVERE SEPSIS and NITRIC OXIDE wever, in abciximab treated patients, fewer major bleeding events, decreased recurrent ischemic The formation of NO from the guanidine nitro- events and better coronary artery patency as as- gen group of L-arginine is catalyzed by nitric oxi- de synthases (NOSs)[309,310]. All the three iso-

Turk J Haematol 2002;19(3):349-389 373 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Fareed J. Antithrombotic Agents in the Management of Sepsis

forms, namely, endothelial cell NOS (ecNOS or c. Induction of iNOS protein and activityin lung NOS III), brain NOS (bNOS or NOS I) and inducible and liver of rats with endotoxic shock (Ruetten NOS (iNOS or NOS II) are inhibited with NG- and Thimmermann). This suggested that preven- monomethyl-L-arginine (L-NMMA). Although the tion of the activation of NFkB by calpain inhibitor I iNOS is absent from mammalian cells under may be useful in the therapy of circulatory shock physiological conditions, it is induced by proinf- in local or systemic inflammation[327]. Dexametha- lammatory stimuli, such as bacterial lipopolysacc- sone which is known to inhibit the endotoxin-me- haride or the cytokines TNF-α, IL-1ß or IFN-c. Un- diated induction of iNOS in vivo and in vitro, also like ecNOS and bNOS, iNOS tightly binds calmo- inhibits the action of transcription factors AP-1 and dulin and hence is not regulated by intracellular NFkB[328,329]. A recent phase III trial utilizing conti- calcium levels and generates large amounts of nuous infusion of L-NAME, a specific inhibitor of NO [311,312]. About 75% of deaths in septic shock, iNOS, was discontinued due to increased adverse occurring within hours and days after the onset of effects such as decreased cardiac output and inc- shock are caused by therapy-resistant hypotensi- reased pulmonary artery pressure and increased on, suggesting that peripheral vascular failure is mortality in the L-NAME group[330]. the key factor that determines the outcome[313]. CONCLUSIONS The other deaths which occurs days or weeks af- ter stabilization of are due to mul- Sepsis continues to be a leading cause of de- tiple organ failure, most commonly the sequence ath in the surgical with morta- of events involves adult respiratory distress lity ranging from 30-80%. Zimmerman et al found syndrome (ARDS), renal and hepatic failure. Inhi- no changes in the incidence of organ failure, of bitors of iNOS activity like NG-cyc lopropyl-L-argi- multiple organ failure and of mortality from 1982- nine, NG-nitro-L-arginine, and its methyl ester, L- 1990[331]. From Hippocrates first description in NAME and L-NMMA reduced the ca- 460 A.D. of what today is called SIRS until today, used by endotoxin in laboratory animals, sugges- a large body of knowledge has been accumula- ting a therapeutic rationale for their use[314-316]. ted. After 2000 years of research on sepsis, the Reducing the enhanced generation of NO by inhi- basic principles of septic conditions are now un- bitors of the iNOS induction and inhibitors of pro- derstood[332]. It is also understood that an identifi- tein kinase C (PKC) or of protein tyrosine kinase able pathogen is not necessarily the trigger of the or of the activation of NFkB[317-321]. Other agents disease, rather the human organism himself plays which inhibit iNOS include glucocorticoids, throm- a key role in the natural history of the disease. In bin, or ethanol; macrophage deactivating factor 1990, Mitchie and Wilmore studied much about and transforming growth factor-ß, platelet-derived TNF-α physiology from an evolutionary point of vi- growth factor. Endothelin-1, IL-4, IL-8, IL-10 and IL- ew[333]. In 1996, R.C. Bone described SIRS as 13[312,322-326]. NFkB, under physiological conditi- the result of dysregulation of the organisms’ biolo- ons is held in an inactive form in the cytoplasm by gical response to certain stimuli[334]. In 1996, Go- the inhibitory protein IkB-α, which prevents its ac- din and Buchman presented the hypothesis of the tivation and translocation to the nucleus to induce multiple organ dysfunction syndrome being the the expression of specific genes. Activation of consequence of biological oscillatory sys- [335] NFkB involves the signal-induced phosphorylati- tems . In the past decade there has been re- on of IkB-α, resulting in its proteolytic degradation markable progress in the field of antithrombotic and the release of NFkB. Proteolytic degradation agents in the management of severe sepsis. En- of IkB-α in vivo by cystein protease inhibitor cal- dothelial damage, activated leukocytes, altered pain inhibitor I and dexamethasone resulted in at- platelet function and hypercoagulability lead to the tenuation of development of septic organ dysfunction. Endo- genous anticoagulants, including antithrombin, a. The circulatory failure, thrombomodulin, protein C, APC and TFPI regu- b. Multiple organ failure and late the function of vascular endothelial cells and neutrophils and also exert an antiinflammatory ef-

374 Turk J Haematol 2002;19(3):349-389 Iqbal O, Tobu M, Hoppensteadt D, Aziz S, Messmore H, Antithrombotic Agents in the Management of Sepsis Fareed J.

fect. Hence, these anticoagulants prevent the sepsis and organ failure and guidelines for the use progression to severe sepsis. F VIIa, F Xa and of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American Col- thrombin directly activate cells, by cleavage of the lege of Chest Physicians/Society of Critical Care [336] cell surface protease activated receptors . F Xa . Chest 1992;101:1644-55. inhibitor DX-9065a modulated the leukocyte en- 2. Angus DC, Line-Zwirble WT, Lidicker J, et al. Epi- dothelial cell interaction in endotoxaemic rats[337]. demiology of severe sepsis in the United States: Leukocytes play an important role in the develop- Analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303-10. ment of sepsis-induced multiple organ dysfuncti- 3. Martin GS, et al. Incidence of sepsis in US continu- on syndrome (MODS). Leukocytes also produce es to rise. (Study findings presented at the 67th an- procoagulant and anticoagulant factors and influ- nual scientific meeting of the American College of ence the coagulation process. They also provide Chest Physicians in Philadelphia, November 7, specific receptors that serve as direct molecular 2001 (2001 MD Consult L.L.C., http://www/mdcon- links between inflammation and coagulation. A re- sult.com by Anthony J. Brown, MD). 4. Bone RC, Grodzin CJ, Balk RA. Sepsis: A new ceptor for F Xa, effector protease receptor 1 hypothesis for pathogenesis of the disease pro- (EPR-1) was expressed on leukocytes and endot- cess. Chest 1997;112:235-43. helium. Xa participates in EPR-1 related leukocyte 5. Rangel-Fraustro MS, Pittet D, Costigan M, Hwang activation and platelet and endothelial cell induced T, Davis CS, Wenzel RP. The natural history of the thrombin formation. EPR-1 signalling is mediated systemic inflammatory response syndrome (SIRS): by F Xa binding and the other requiring active si- A prospective study. JAMA 1995;273:117-23. te. F Xa also activates cells by an EPR-1 indepen- 6. Annane D, Sebille V, Troche G, Raphael JC, Gajdos P, Bellisant E. A 3-level prognostic classification in dent fashion[338]. On the endothelium, F Xa elicits septic shock based on cortisol levels and cortisol expression of IL-6, IL-8 and monocyte chemotac- response to corticotropin. JAMA 2000;283:1038- tic protein-1 by an active-site independent reacti- 45. on, independent of EPR-1[339-341]. Increased exp- 7. Angus DC, Birmingham MC, Balk RA, et al. E5 mu- ression of E-selectin, ICAM-1, and VCAM-1 ac- rine monoclonal antiendotoxin antibody in gram-ne- companies leukocyte adhesion through enzyma- gative sepsis: A randomized controlled trial. JAMA 2000;283:1723-30. tic activation[342]. High doses of DX 9065a appe- 8. Increase in National Hospital Discharge Survey ra- ared to be beneficial in septic MODS[337]. An early tes for septicaemia- United States, 1979-1987. JA- phase II trial of DX-9065a in severe sepsis is on- MA 1990;263:937-8. going in Japan. A recent study to analyze the inf- 9. Linde-Zwirbe WT, Angus DC, Carcillo J, Lidicker J, luence of heparins (UFH and certoparin, a LMWH) Clermont G, Pinsky MR. Age-specific incidence on the generation of cytokines with known antiinf- and outcome of sepsis in the US. Crit Care Med lammatory activities (IL-1ra, IL-6, IL-10) and of IL- 1999;27:(Suppl 1):A33 (A). 12p40 from human leukocyte fractions concluded 10. Dubois MJ, Vincent JL. New hope for sepsis. Clini- that certoparin caused a pronounced response on cal Researcher 2001; Vol No 3. IL-6 generation when compared to UFH, at same 11. Camerota A, Creasey A, Patla V, Larkin V, Fink M. Delayed treatment with recombinant human tissue concentrations[343]. IL-6 has been recently repor- factor pathway inhibitor improves survival in rabbits [344] ted to be heparin-binding protein . LMWHs with gram-negative peritonitis. J Infect Dis 1998; (Dalteparin) inhibited TNF-α-induced leukocyte 177:668-76. rolling along microvascular endothelium, expla- 12. Park T, Creasey A, Wright S. Tissue factor pathway ining their antiinflammatory effects at a dose of inhibitor blocks cellular effects of endotoxin by bin- 5000 units/kg, a relatively high dose when compa- ding to endotoxin and interfering with transfer to CD14. Blood 1997;89:4268-74. red to 200 units/kg for deep vein thrombosis[345]. 13. Broze GL, Warren LA, Novotny WF, et al. The lipop- Footnote: This article is dedicated to Profes- rotein-associated coagulation inhibitor that inhibits sor Orhan N. Ulutin, MD. the factor VII-tissue factor complex also inhibits fac- tor Xa. Insight into the possible mechanism of acti- REFERENCES on. Blood 1988;71:335-43. 14. Fareed J, Callas D, Hoppensteadt D, Walenga J. 1. Bone RC, Balk RA, Cerra FB, et al. Definitions for Modulation of endothelium by heparin and related

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Address for Correspondence: Omer IQBAL, MD Loyola University Medical Center Maywood, Illinois-60153, USA

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